Brucella melitensis, an intracellular human pathogen, is largely controlled by CD8+ T cells, but the Brucella proteins containing peptides recognized by protective CD8+ T cells remain unknown. Our goal is to identify specific Brucella CD8+ T cell epitopes and test their ability to protect mice against infection with virulent Brucella. To do this we will first isolate and identify naturally processed and presented Brucella-specific T cell epitopes bound to Major Histocompatibility Complex class I (MHC I) molecules on infected macrophages. T cell epitopes will be identified using Mass Spectrometry (MS) and bioinformatics to correlate an epitope with the respective Brucella protein. Second, we will assess CTL activity against the Brucella antigens using an invasive E. coli vector to immunize mice. Third, we will assess the impact of antigenic Brucella proteins and T cell epitopes on clearance of virulent Brucella from mice. We are confident of achieving these goals based on preliminary data that demonstrate our ability in each specific aim. Also, we have shown significant CTL response to antigen from mice immunized with the recombinant E.coli vectors. We believe our goal of developing and testing this novel approach to elucidate CD8+ CTL epitopes and their contributions to clearance of Brucella in vivo will fill the void in identifying Brucella proteins important for clearance of this intracellular bacterium by cytotoxic CD8+ T cells. Our specific aims are: 1. Identify naturally processed and MHC I presented Brucella peptides. Mouse macrophage RAW264.7 cells will be infected with Brucella melitensis. MHC I-Brucella peptide complex will be co- immunoprecipitated from the cell membrane and the peptide purified. Peptides will then be subject to LC-MS, and data will be analyzed for Brucella T cell epitopes. Affinity of Brucella peptide binding to MHC I molecules will be used to confirm the identify of high binding peptides. 2. Assess CTL activity against Brucella proteins containing specific T cell epitopes. Invasive E. coli vectors will be engineered to express Brucella proteins containing T cell epitopes. These recombinant E. coli vectors will be used to immunize mice. H-2 haplotype matched cell lines expressing the Brucella peptides will be used as stimulator/target cells for CTL assays of splenic T-cells isolated from the immunized mice. CTL data analysis will identify Brucella proteins containing CD8+ T cell epitopes important for clearance of intracellular Brucella. 3. Evaluate Brucella proteins delivered by invasive E. coli vectors to induce CTL activity and host protection. Clearance of luxCDABE-expressing virulent B. melitensis will be determined in mice immunized with invasive E. coli vectors expressing Brucella proteins and clearance will be correlated to CTL activity. Adoptive transfer of Brucella specific CD8+ T cells to naive recipient mice will confirm the effectiveness of different Brucella-specific CTLs to clear virulent B. melitensis infection. Thus, this aim will link CTL activity to biologic relevance in clearing Brucella infection. Identifying Brucella proteins that contain CD8+ T cell epitopes would be crucial to understanding the contribution of the Brucella proteome to immune recognition and host protection. Project Narrative: The proposal will identify specific B. melitensis CD8+ T cell epitopes and test their ability to protect mice against infection with virulent Brucella. We will first isolate and identify naturally processed and presented Brucella-specific T cell epitopes bound to Major Histocompatibility Complex class I (MHC I) molecules on infected macrophages. T cell epitopes will be identified using Mass Spectrometry (MS) and bioinformatics to correlate an epitope with the respective Brucella protein. Second, we will assess CTL activity against the Brucella antigens using an invasive E. coli vector to immunize mice. Third, we will assess the impact of mice immunized with antigenic Brucella proteins containing T cell epitopes to clear virulent Brucella from mice using in vivo biophotonic imaging. We are confident of achieving these goals based on preliminary data that demonstrate our ability in each specific aim. The Problem: Brucella melitensis, a Gram-negative facultative intracellular bacterium, induces chronic infectious disease in humans and domestic animals. Immunological protection against intracellular Brucella appears to be mediated by cytotoxic CD8+ T cells, but the peptides recognized by protective cytotoxic T cells remain unknown. Identifying Brucella proteins that contain CD8+ T cell epitopes would be crucial to understanding the contribution of the Brucella proteome to immune recognition. What Brucella genes or proteins should encompass an effective, safe Brucella vaccine for humans is poorly understood. The Product: Our goal of developing and testing this novel approach to elucidate CD8+ CTL epitopes and their contributions to clearance of Brucella in vivo will fill the void in identifying Brucella proteins important for clearance of this intracellular bacterium by cytotoxic CD8+ T cells. The invasive, non- pathogenic E. coli vector can stimulate a potent CTL response providing a strategy for Brucella immunization to test identified MHC I associated epitopes for CD8+ T cell protective immunization. Identifying B. meltinesis peptides that confer protection and determining the mechanisms that initiate host protection will permit designing vaccine vector delivery systems for host protection.